Texas red egf

Antibodies against Notch1 ICD (D1E11, 1:100 IF, 1:1,000 WB), Notch1 V1744 (D3B8, 1:500 WB), EGFR (D38B1, 1:100 IF, 1:1,000 WB), pEGFR (D7A5, 1:1,000 WB), GFP (D5.1, 1:1,000 WB), non-phospho (active) β-catenin (D13A1, 1:1,000 WB), GAPDH (14C10, 1:10,000 WB), and YAP (D8H1X, 1:200 IF) were from Cell Signaling Technologies. β-catenin antibody (14, 1:1,000 WB) was from BD Biosciences. E-cadherin antibody (HECD-1, 1:1,000 IF, 1:1,000 WB) was from Takara Bio. Notch1 ECD (ABS90, 1:1,000) was from Millipore. FAM83H antibody (1:1,000 WB) was from Bethyl Laboratories. Lamin B1 antibody (12987-1-AP, 1:1,000) was from Proteintech. TexasRed-EGF, rhodamine phalloidin, and Alexa Fluor 488, 568, and 647 goat anti-mouse and anti-rabbit IgG secondary antibodies (1:400) were from Invitrogen. Alexa Fluor 647 azide and EdU cell proliferation kit were from Invitrogen. Hoescht and DAPT were from Sigma. Anti-SNAP (P9310S, 1:1,000), SNAP-Capture, and SNAP-Surface 488 were from New England Biolabs.

Cells were incubated with 2 μg/ml of Texas Red‐EGF (Invitrogen) for 10 min at 37°C and washed twice with cold modified PBS (PBS pH7.4, 0.5 mM MgCl₂, 0.5 mM CaCl₂, 5 mM glucose, 0.2%BSA), three times with cold citrate buffer pH 4.6 (25.5 mM citric acid, 24.5 mM sodium citrate, 280 mM sucrose), and re‐equilibrated with two PBS washes. Cells were returned to 37°C serum‐free media. At the appropriate time point, cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence as described below.